Unit 5. Microscopy & Staining

RESOLVING POWER

closest distance between two objects that when magnified still allows the two objects to be distinguished from each other

False

Shorter the wavelength of light used in the instrument, the greater the resolution

T or F

the longer the wavelength of light is used in the instrument, the greater the resolution

Refractive Index

measure of the light-bending ability of a medium

CONTRAST

needed to make objects stand out from the background

CONTRAST

achieved by staining techniques that highlight organisms and allow them to be differentiated from one another and from background material and debris

Kohler Illumination

designed to provide maximum illumination and resolution when observing images using a microscope

higher ; higher

the _______ the objective magnification; the ________ the light intensity needed, and vice versa

PHASE-CONTRAST MICROSCOPY

microscope used for detailed examination of internal structures in living microorganisms →not necessary to fix or stain the specimen

Reinforcement (relative brightness)

wave peak of light rays from one source coincides with the wave peak of light rays from another source

Interference (relative darkness)

wave peak from one light source coincides with the wave trough from another light source

Diffraction

scattering of light rays as they touch a specimen's edge

1. direct from light source

2. reflected or diffracted from a particular structure in the specimen

ENUMERATE

set of light rays

FLUORESCENT MICROSCOPY

A microscopy technique that uses fluorochromes to produce visible light after excitation by ultraviolet light.

Fluors or Fluorochromes

A fluorescent dye that absorbs ultraviolet light and emits visible fluorescent light.

Excitation filter

A filter that allows only the wavelength needed to excite the fluorochrome to pass through.

Barrier filter

a filter that prevents the excitation wavelengths from damaging the eyes of the observer

• Acridine Orange (for nucleic acid)

• Auramine (for mycolic acid)

• Fluorescein Isothiocyanate (FITC)

these dyes require BLUE excitation light

Calcofluor White

these dyes require VIOLET excitation light

exciter filter: 450-490 wavelength

barrier filter: 515 wavelength

Acridine Orange, Auramine, Fluorescein Isothiocyanate (FITC)

exciter filter:

barrier filter:

exciter filter: 355-425 wavelength

barrier filter: 460 wavelength

Calcofluor White

exciter filter:

barrier filter:

FLUOROCHROMING

direct chemical interaction between the fluorescent dye and a component of the bacterial cell

ACRIDINE ORANGE

this dye binds to nucleic acids (nonspecific) and is used to confirm the presence of bacteria in blood culture. it emits the color BRIGHT ORANGE.

False

T or F

acridine orange can differentiate G (-) and G (+) bacteria

ACRIDINE ORANGE

the dye used for detection of cell wall–deficient bacteria grown in culture

AURAMINE-RHODAMINE

this dye have affinity to waxy mycolic acids (non-specific) in the cell walls of mycobacteria. appear BRIGHT YELLOW or ORANGE against a greenish background

AURAMINE-RHODAMINE

the dye used for initial characterization of cells grown in culture

AURAMINE-RHODAMINE

used to enhance detection of mycobacteria directly in patient specimens

CALCOFLUOR WHITE

this dye bind in the cell walls of fungi. this directly detect fungi in clinical material and observe subtle characteristics of fungi grown in culture

CALCOFLUOR WHITE

this dye visualize some parasites such as microsporidia

IMMUNOFLUORESCENCE

this microscopy technique uses antibodies that are conjugated to a fluorescent dye. the dye-antibody conjugate detect, or “tag,” specific microbial agents. thus making microorganisms become readily detectable by a fluorescent microscopy

IMMUNOFLUORESCENCE

combines the amplified contrast provided by fluorescence with the specificity of antibody- antigen binding

• Legionella spp.,

• Bordetella pertussis,

• Chlamydia trachomatis

what are the bacteria detected by IMMUNOFLUORESCENCE

APPLE GREEN

FITC emits what color

DARK-FIELD MICROSCOPY

what microscopy technique is used to detect SPIROCHETES

DARK-FIELD MICROSCOPY

involves the alternation of microscopic technique rather than the use of dyes or stains to achieve contrast

DARK-FIELD MICROSCOPY

condenser does not allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle

ELECTRON MICROSCOPY

uses electrons instead of light to visualize small objects

TRANSMISSION ELECTRON MICROSCOPE (TEM)

passes the electron beam through objects and allows visualization of internal structures

SCANNING ELECTRON MICROSCOPE (SEM)

uses electron beams to scan the surface of objects and provides three-dimensional views of surface structures

1. Observe and appreciate the appearance of microorganism

2. Differentiate one microorganism or group of microorganism from another

3. Identification of microorganisms and their special structures

ENUMERATE

purpose of staining

positive

what is the charge of basic dyes?

negative

what is the charge of acidic dyes?

b. negatively

cationic dyes bind to _____________ charged molecules

a. positively

b. negatively

a. positively

anionic dyes bind to _____________ charged molecules

a. positively

b. negatively

• Crystal Violet,

• Methylene Blue

• Malachite Green

• Safranin

ex. of BASIC DYES

• Eosin,

• Acid Fuchsin

• Nigrosin

ex. of ACIDIC DYES

7

Bacteria are slightly negatively charged at pH __

FIXING

This kills the microorganisms and fixes them to the slide and preserves various parts of microbes in their natural state with only minimal distortion.

• Heat-fixed

• Methanol Fixation

ex. of FIXING

95% ; 1

methanol fixation uses ____ methanol for ___ minute

Methanol Fixation

this fixation technique preserves morphology of host cells, bacteria especially useful for examining bloody specimen material

1. SIMPLE

2. DIFFERENTIAL

3. SPECIAL

ENUMERATE

Three kinds of staining techniques

SIMPLE STAINS

single stain is used to highlight the entire microorganism so that cellular shapes and basic structures are visible stain is applied to the fixed smear for a certain length of time and then washed off, dried and examined

• Methylene Blue

• Carbolfuchsin

• Safranin

• Crystal Violet

ex of SIMPLE STAIN

DIFFERENTIAL STAINS

react differently with different kinds of bacteria and thus can be used to distinguish them

• Gram Stain

• Acid-Fast Stain

ex of DIFFERENTIAL STAINS

True

T or F

Gram-positive cells retain the dye and remain purple

False

(Gram-negative cells do not retain the dye)

T or F

Gram-negative cells retain the dye

Mordant

chemically bond the alkaline dye to the bacterial cell wall

GRAM’S IODINE

ex of MORDANT

Staphylococcus aureus

QC used for G (+)

Escherichia coli

QC used for G (-)

Crystal Violet–Iodine (CV-I) complex.

what do you call the complex formed in the peptidoglycan when Crystal Violet and Gram’s Iodine bind

Dark Purple to Deep Blue

color or G (+)

safranin

counterstain used in G staining

Pink to Deep Magenta

color of G (-)

False

(decolorizer dehydrates the outer cellular membrane, leaving holes in the membrane and effectively washing or removing the CV-I complex from the cells)

T or F

CV-I complex remains in the peptidoglycan layer of G (-) bacteria even after decolorizing

• Neisseria,

• Veilonella

• Branhamella (Moraxella)

All COCCI are GRAM-POSITIVE except

• Arcanobacterium

• Actinomyctes,

• Bacillus,

• Clostridium,

• Corynebacterium,

• Erysipelothrix,

• Eubactrium,

• Gordonia,

• Kuthria,

• Lactobacilli,

• Listeria,

• Mycobacteria,

• Nocardia,

• Propionibacterium

• Tsukamurella

All BACILLI are GRAM-NEGATIVE except

NEGATIVE

All spirochetes are GRAM-____________

1. Removal of MgRNA

2. Autolysis

3. Acidic solution of Gram’s Iodine

4. Technical error Over-decolorization

ENUMERATE

reasons why G (+) becomes G (-)

precipitation with bile salts

what is the reason for removal of MgRNA

ACID-FAST STAIN

They binds strongly only to bacteria that have a waxy material in their cell walls

Carbolfuchsin

primary stain if AFS

Heat or Tergitol

allow the stain to penetrate into the waxy surface of acid-fast microorganisms

3% acid alcohol (ethanol or HCL)

removes excess stain (decolorizer) in AFS

Methylene Blue or Malachite Green

secondary stain used in AFS

PINK (RED)

Acid-Fast Organisms yield the color

DARK BLUE

Nonacid-Fast Organisms yield the color

BLUE to BLUE- GREEN

in AFS background material should stain _______________________

Mycobacterium leprae

Hansen’s bacillus ; causes leprosy

1. Ziehl-Neelsen (Hot Staining Method)

2. Kinyoun’s Method (Cold Staining Method)

3. Pappenheim Method

4. Baumgarten Method

5. Auramine-Rhodamine Method

[Zelle, k p ba?]

ENUMERATE

AFS methods

heating or steaming process

5-7 mins

WAYS TO FACILITATE ACID-FAST STAINING

• Use of _____________________ for ________

  to temporarily remove the mycolic acid, while the

  smear is flooded with stain

dye and phenol

WAYS TO FACILITATE ACID-FAST STAINING

• Increasing the concentration of _____________ in

  the staining reagent

TERGITOL

WAYS TO FACILITATE ACID-FAST STAINING

• Addition of a wetting agent like ___________

MYCOLIC ACID

Acid-fast organism contain ___________ in their

outer membrane, making the cells waxy and resistant

to staining with aqueous based stains such as the

Gram stain

Pappenheim Method

AFS method used to differentiate M. smegmatis from M. tuberculosis

• Mycobacterium tuberculosis (RED)

• Mycobacterium smegmatis (BLUE)

Pappenheim Method is used to differentiate what bacteria?

rosolic acid and alcohol

M. smegmatis is decolorized by the mixture of ____________________ coloring it BLUE

Baumgarten Method

AFS method that is used to differentiate M. tuberculosis from M.leprae

Mycobacterium tuberculosis (BLUE) ; Mycobacterium leprae (RED)

Baumgarten Method differentiates _______________ from _________________

• Pappenheim Method - BLUE

• Baumgarten Method - RED

M. tuberculosis yield what color in

• Pappenheim Method

• Baumgarten Method

Auramine-Rhodamine Method

AFS method that is selective for the cell wall of AFB

Ziehl-Neelsen method

what method is ideal for concentrated smears partially acid-fast bacilli---Nocardia spp

Hydrochloric Acid and Ethanol

Acid-alcohol is composed of?

• colonial age,

• medium for growth

• UV light

Acid-Fastness is affected by?

SPECIAL STAINS

These are used to color and isolate specific parts of microorganisms endospores and flagella, and reveal the presence of capsules

CELL WALL STAIN

Dyar Method

INDIRECT/NEGATIVE STAINING

colorless bacteria against a colored background excellent technique for studying bacterial vacuoles and viral morphology

india ink

dye used in INDIRECT STAINING